We plan to study regulatory events important in the control of metabolic pathways in normal and neoplastic cells. Two enzymes, situated at branch points in divergent biosynthetic pathways, will be purified and characterized, and the biochemical and physiological processes by which each is regulated isolated and studied at the cellular and molecular levels. Normal and tumor tissue will be compared, to define possible metabolic defects in tumor tissues, and to outline important sites at which anti-neoplastic therapy might be directed. The specific studies include: (a) purification and properties of rat liver glutamine synthetase; this enzyme is subject to feedback inhibition, and different tissue levels of activity are found under various conditions, including in developing animals, in tumors, and after hormone adminstration. Product, and feedback inhibition, as well as the synthetic, and degradative rates of enzyme protein, as they are modified in response to various stresses, and in malignancy, will be studied. Where applicable, the studies will be applied in mammalian cell culture systems: (b) purification and characterization of rat liver 5-phosphoribosyl-alpha-1- pyrophosphate (PRPP) synthetase (in collaboration with Dr. Daniel G. Roth). PRPP is a precursor for purines (de novo and salvage pathways), pyrimidines, and pyridine nucleotides, as well as for histidine and tryptophan biosynthesis. The regulation of PRPP synthetase will be studied, correlating biochemical control with changing cellular needs for PRPP. Enzyme from developing animals, after partial hepatectomy, after administration of drugs, and in rapidly growing tumors will be studied. The methods used will be fractionation techniques for proteins, as well as standard enzymological and protein chemical approached to an analysis of activity and structure of enzymes. Antibodies to enzyme protein will be used with radioactive labeling to determine synthetic and degradative rates; factors important to regulation will be isolated and explored also by these methods. The results of this project should clarify some aspects of overall cellular regulation, particularly as related neoplasia.